LA NUTRIGENOMICA NEL DIFFERENZIAMENTO MUSCOLARE SCHELETRICO: RUOLO DEL RESVERATROLO NEL PROCESSO DI MIOGENESI ED IPERTROFIA IN MIOBLASTI MURINI.

ABSTRACT Nutrigenomics in musculoskeletal differentiation: Resveratrol action on myogenesis process and hypertrophy induction in C2C12 myoblasts The new era of nutrition research translates empirical knowledge to evidence-based molecular science, because food components interact with our body at system, organ, cellular and molecular level. Modern nutrition research focuses on promoting health, preventing or delaying the onset of disease, optimizing performance and assessing risk. Nutrigenomics studies elucidate the ability of bioactive food components to influence gene expression pattern, protein synthesis, degradation and post-translational modifications. Understanding the interrelationship among genome function, cellular metabolism processes and dietary components will enable precise realization of the “Personalized Nutrition” to optimize individual health, diagnosis and nutritional treatment of several chronic diseases, and perhaps increase human longevity. Regulation of skeletal muscle formation (myogenesis) is essential for normal development as well as to prevent pathological conditions such as muscular dystrophies and inflammatory myopathies. Skeletal muscle differentiation is a dynamic multistep process that involves two simultaneous phenomena. The first is the induction of muscle-specific genes expression by Myogenic Regulatory Factors (MRFs), such as Myf-5, MyoD, Myf-6 and Myogenin. Second phase is the commitment of myogenic cell into skeletal muscle: mononucleated undifferentiated myoblasts break free from the cell cycle, elongate and fuse into multinucleated myotubes. Furthermore, cytoskeletal reorganization and severe activation of specific protein kinases represent key regulator mechanisms of skeletal muscle metabolism. Skeletal muscle hypertrophy can be defined as an overall augmentation in muscle mass, as a result of an increase in the size of pre-existing skeletal muscle fibers accompanied by enhanced protein synthesis without an apparent increase in the number of myofibers. Many studies have established that Insulin Like Growth Factor 1 (IGF-1) strongly activates muscle hypertrophy by stimulating the PI3-K/AKT kinases pathway. AKT, in turn, activates the downstream kinase mTOR, which stimulates p70 S6 kinase and other effectors, ultimately culminating in enhancing protein synthesis. Resveratrol (RSV), natural polyphenol found in grapes and in other fruits, has a plethora of health benefits in a variety of human diseases: cardio and neuroprotection, immune regulation, cancer chemoprevention, DNA repair, activation of Sirtuins (SIRT1), prevention of mitochondrial disorder, avoidance of obesity-related disease. In skeletal muscle, RSV acts on protein catabolism and muscle function, conferring resistance against oxidative stress, injury and cell death, but its action mechanisms and protein targets are not completely known. To elucidate the underlying mechanism of RSV action, much research has been focused on different tissues and cell types, but less attention has been given to its effect on myogenesis. Aim of this work was the study of RSV effects on cell cycle regulation, differentiation process and hypertrophy genesis in C2C12 murine immortalized cell line, a good in vitro model for the major step of myoblasts proliferation and differentiation. In detail, mouse myoblast C2C12 immortalized cell line is a sbuclone of C2 myoblasts, which spontaneously fuse and differentiate into multinucleated myotubes as a result of both the achievement of myoblast confluence and the removal of the serum growth factors. After preliminary dose/response experiments, performed in order to determine the effective RSV dose able to induce biological response in C2C12 cells, two different concentrations of this natural compound was chosen: 0,1 and 25 μM. Cells were incubated in growth medium with/without RSV (0.1 or 25 μM) for 24, 48, 72 hours to study proliferative phase. To examine differentiation phases and hypertrophy induction, at 70% of confluence, cells were maintained in growth medium or transferred in differentiation medium both with/without RSV (0.1 or 25 μM) for 24, 48, 72, 96 hours. Results showed that RSV could regulate C2C12 myoblasts growth capacity. This effect were visible not only in the kinetics of cell growth, but also in the morphological changes. Data obtained from gene and protein expression studies showed how RSV can be able to act on cell cycle regulation to induce differentiation. Also RSV could control MRFs expression and muscle-specific proteins synthesis during myogenesis progression. In late differentiation, we evaluated the positive action of RSV on hypertrophy: RSV increased AMPK and IGF-1 proteins content and induced hypertrophic morphological changes in neo-formed myotubes, modulating cytoskeletal proteins expression. In summary, these data demonstrate that RSV could be able to control proliferation, start myogenesis process and induce hypertrophy. RSV seems to be able of regulating cell cycle progression, following cell cycle arrest and early induction of differentiation, through its action on the expression of specific cell cycle regulators, myogenic regulatory factors and muscle-specific structural proteins. This work may constitute novel proof of principle to potential use as integrator/drug for this nutrient compound in the treatment of clinical conditions characterized by chronic functional and morphological muscle impairment.